1. Field of the Invention
The present invention relates generally to electrochemical sensors that can be used for quantification of a specific component or analyte in a liquid sample. Particularly, the present invention relates to an electrochemical-based sensor for measuring bilirubin in biological fluids.
2. Description of the Prior Art
Bilirubin is the main bile pigment that is formed from the breakdown of heme from red blood cells. The broken-down heme travels to the liver, where it is secreted into the bile by the liver. It is estimated that approximately 200 to 300 milligrams of bilirubin and its derivatives are formed each day in a normal human adult by degradation of hemoglobin within the liver, spleen, and bone marrow. Normally, a small amount of bilirubin circulates in the blood. Serum bilirubin is considered a true test of liver function as it reflects the liver's ability to take up, process, and secrete bilirubin into the bile.
Two fractions of bilirubin are present in blood serum. One is free or non-conjugated bilirubin and the other conjugated bilirubin since it has become conjugated with glucuronic acid and rendered water-soluble. The diagnostic significance of bilirubin is well established. An excessive amount of bilirubin within the human body, referred to as jaundice, is recognized as evidence of a variety of disease conditions, particularly diseases of the liver. In an adult, bilirubin could be excreted from the body by way of the bile fluids. In a fetus, bilirubin can easily cross the placenta and be removed through the mother's liver. Newborn infants and especially prematurel infants, however, are particularly susceptible to hyperbilirubinemia. Hyperbilirubinemia is defined as an abnormally high level of bilirubin in the blood, manifested by jaundice, anorexia, and malaise, occurring in association with liver disease and certain hemolytic anemias. Because an infant's liver is not fully functioning for the first few weeks after birth, bilirubin may accumulate above normal adult levels, which “stains” infant's skin leading to jaundice. Clinically, untreated jaundice in neonates can lead to impaired learning ability, mental retardation, cerebral palsy, seizures, deafness, or even death.
There are several methods used to determine bilirubin concentration. The leading method is the so-called diazo method, which measures the purplish-red color of azobilirubin formed by the reaction of bilirubin with a diazo compound. There are numerous versions of the diazo method, differing in the reaction conditions and reagent composition. The method according to Jendrassik and Graf has been recommended by the National Committee for Clinical Laboratory Standard (NCCLS) as the procedure of choice for total bilirubin measurement. There are, however, disadvantages associated with this method. For example, the reagent solutions for the Jendrassik/Gróf method can be used for only about 5 days after their preparation, and the measured values are affected by L-ascorbic acid and hemoglobin, which are present in a sample.
The total bilirubin level in bodily fluids has been measured by direct spectrophotometric estimation at 454 nm with correction at 540 nm. This direct spectrophotometric measurement has been improved by making observations before and after the destruction of bilirubin with the enzyme bilirubin oxidase. O'Leary et. al (Ann. Clin. Biochem., 30:175-179, (1993)) describes a two-step method based on the measurement of absorbance of bilirubin itself at about 480 nm, followed by measurement of absorbance at 480 nm after destruction of the bilirubin with ferricyanide. This method is limited due to the use of the 480 nm wavelengths of detection. At this wavelength, highly lipemic or hemolyzed sera may cause the absorbance, and a false result may be produced.
U.S. Pat. No. 6,326,208 (2001, Denney) discloses an assay for measuring total and direct bilirubin colorimetrically that includes the use of a first or a second reagent, respectively. For total biliruben, the first reagent includes a solvent in which the bilirubin and the proteins are soluble, an acid, and a promoter for speeding up the oxidation of the bilirubin and the formation of a chromophore that is detectable in the near-infrared region of the spectrum. Useful promoters are nitrite ion, cupric ion and ferricyanide ion. For direct bilirubin, the second reagent includes an aqueous component containing an acid for dissolving both the serum or plasma proteins and the conjugated bilirubin of the sample, and an oxidant for promoting the oxidation of the conjugated bilirubin while promoting very little oxidation of unconjugated bilirubin. The oxidant should have a low single electrode potential between 0.34 and 0.78 volts. Useful oxidants are cupric ion, ferric ion and ferricyanide ion.
U.S. Pat. No. 5,563,072 (1996, Tokuda et al.) discloses a method of measuring direct bilirubin colorimetrically that includes using bilirubin oxidase or a chemical oxidizing agent of vanadic ions, manganese (III) ions, ferricyanide ions or copper ions. Another method includes additionally using a reaction inhibitor that is a water-soluble high polymer or a nonionic surfactant having a HLB value of 15 or more.
Yet another method involves the use of a dry film for determination of serum bilirubin, which is based on the observation that bilirubin interacts with certain cationic polymers (mordants) giving a unique spectral change. A transparent support is coated with a buffered gelatin-mordant layer and an isotropically porous polymer spreading layer. The spreading layer contains all of the necessary components (diazonium salt, accelerator, surfactant) for the quantification of bilirubin. The buffered gelatin-mordant layer maintains the pH of the dry film, minimizes the dependence of spot size on protein concentration, and increases the sensitivity of the dry film. The dry film, however, is only stable for 4 weeks at room temperature and cannot be used for whole blood analysis.
Therefore, what is needed is a bilirubin sensor that is usable with whole blood. What is also needed is a bilirubin sensor that is capable of performing an accurate and sensitive analysis of the concentration of bilirubin in a small volume of biological sample. What is further needed is a bilirubin sensor that is disposable.